eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Alignment of the optical path between the CMOS (complementary metal oxide semiconductor) camera and the individual or branching patch cord
<ol>
	<li>Loosen all screws on the 5-axis translator (Figure 1B).</li>
	<li>Screw in the patch cord (Figure 1B) to the adaptor [SMA (sub-miniature A) or FC (fiber optic connector)] that is affixed to the 5-axis translator.</li>
	<li>Turn on the 470 nm excitation light (Figure 1B) at low power (100 &#956;W) and place the tip of the patchcord pointing to an autofluorescent plastic slide. This does not have any bearing on future recordings but is solely for visualizing the alignment process.</li>
	<li>Record from the CMOS camera (Figure 1B) in live mode. Increase the gain or adjust the lookup table (LUT) until the image is not entirely black. The point is to be able to see an image at the focal point of the objective (Figure 1B).</li>
	<li>Advance the 5-axis translator towards the objective, ensuring that the 470 nm light is centered on the fiber at the SMA or FC end of the patch cord until an image can be resolved on the camera.</li>
	<li>Adjust the X and Y axes until the image is centered and well-resolved.</li>
	<li>Visualize the light emitted from the ferrule end of the patch cord. It should appear as an isotropic circle. If a branching patch cord is used, the amount of light emitted at the ferrule ends of each patch cord should be similar. If the circle is not isotropic or the emitted light is unequal, adjust the 5-axis translator in the X-Y axis.</li>
</ol>
2. Setup of ROIs around fibers for measurement of mean fluorescent intensity
<ol>
	<li>Turn on all the excitation lights to visualize the fibers better. Adjust the camera gain so that no pixels are saturated and a clear image of the fibers is present.</li>
	<li>Live record or take a preliminary image.</li>
	<li>Draw ROIs around the fibers and keep them for measuring the mean intensity values during recordings (Figure 1A).</li>
	<li>For multiple fiber recordings, test for independence in signals.
	<ol>
		<li>Live record from all fibers.</li>
		<li>Point one fiber towards a light source and tap with a finger. Large fluctuations should occur solely in that channel (acceptable leakage 1:1000).</li>
		<li>If the signals are not independent, redraw more conservative ROIs and repeat the independence test.</li>
	</ol>
	</li>
	<li>To label and track which ROI corresponds to which fiber, colored tape, or nail polish can be applied to the end of the fibers. Take a picture before the start of any experiment as a secondary reminder.</li>
</ol>
3. Setup of recording arena
<ol>
	<li>Hang the patch cord above the arena using stands, clamps, or holders.</li>
	<li>Make sure that the animal can freely move throughout the entire arena, uninhibited by the length of the fiber.</li>
	<li>If an operant box or open field is used, ensure the patch cord can reach the animal with minimal bending. If this requires a nose poke, ensure that there is enough room overhead to prevent the bending of the fiber. Avoid any excessive bending or twisting of the patch cord.</li>
</ol>
4. In vivo recordings
<ol>
	<li>Visually inspect the distal end of the fibers of the patch cord by eye and with a minifiber microscope. If the surface of the fibers is scratched, repolish the fibers using fiber polishing/lapping film with fine grit (1 &#956;m and 0.3 &#956;m).</li>
	<li>Clean the distal ends of the patch cord with 70% ethanol and a cotton tip applicator.</li>
	<li>Clean the fiber-optic cannulas using 70% ethanol and a cotton tip applicator.</li>
	<li>Connect the ferrule end of the patch cord to the implanted fiber using a ceramic split sleeve covered with a black shrink tube. Ensure the sleeve is tight during the connection. Otherwise, use a new sleeve. 
	NOTE: If there is any space between the patch cord ferrule and the implant, there will be a large amount of signal loss, and the recordings will not work.</li>
	<li>Allow the animal to recover for a few minutes before the start of behavioral testing.</li>
	<li>Start recording the optical signal and run the experiment.</li>
	<li>While recording, keep a careful eye on the live-trace to ensure quality recordings. The signal is expected to rapidly decrease as a function of time in the first 2 min of recording. This effect is caused by heat-mediated LED decay, whereby the increase in heat increases the resistance of the optical element.</li>
	<li>If a jump in the signal that exceeds the on/off kinetics of GCaMP occurs, this often indicates that the sleeve is not tight enough and the space between the patch cord and the implant is changing. In this case, stop the experiment and reconnect the animal using a new sleeve.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S100</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1/2&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S050</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 3/8&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S038</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;m, 0.50 NA, SMA-SMA 
			&#160;Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M59L01</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Optical Post</td>
			<td>Thorlabs</td>
			<td>TR30/M</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Pedestal Post Holder</td>
			<td>Thorlabs</td>
			<td>PH20EM</td>
			<td></td>
		</tr>
		<tr>
			<td>15 V, 2.4 A Power Supply Unit with 
			&#160;3.5 mm Jack Connector for T-Cube</td>
			<td>Thorlabs</td>
			<td>KPS101</td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm Cage Cube with Dichroic Filter Mount</td>
			<td>Thorlabs</td>
			<td>CM1-DCH/M</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_405</td>
			<td></td>
		</tr>
		<tr>
			<td>410 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB410-10</td>
			<td></td>
		</tr>
		<tr>
			<td>465 nm. LED</td>
			<td>Doric Lenses</td>
			<td>CLED_465</td>
			<td></td>
		</tr>
		<tr>
			<td>470 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB470-10</td>
			<td></td>
		</tr>
		<tr>
			<td>560 nm bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-560/14-25</td>
			<td></td>
		</tr>
		<tr>
			<td>560 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_560</td>
			<td></td>
		</tr>
		<tr>
			<td>5-axis kinematic Mount</td>
			<td>Thorlabs</td>
			<td>K5X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Achromatic Doublet</td>
			<td>Thorlabs</td>
			<td>AC254-035-A-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptor for 405 collimator</td>
			<td>Thorlabs</td>
			<td>AD11F</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptor for ajustable collimator</td>
			<td>Thorlabs</td>
			<td>AD127-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Breadboard</td>
			<td>Thorlabs</td>
			<td>MB1824</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping Fork</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td></td>
		</tr>
		<tr>
			<td>Cube connector</td>
			<td>Thorlabs</td>
			<td>CM1-CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual 493/574 dichroic</td>
			<td>Semrock</td>
			<td>FF493/574-Di01-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission filter for GCaMP6</td>
			<td>Semrock</td>
			<td>FF01-535/22-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Enclosure with Black Hardboard Panels</td>
			<td>Thorlabs</td>
			<td>XE25C9</td>
			<td></td>
		</tr>
		<tr>
			<td>Externally SM1-Threaded End Cap for Machining</td>
			<td>Thorlabs</td>
			<td>SM1CP2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-change SM1 Lens Tube Filter Holder</td>
			<td>Thorlabs</td>
			<td>SM1QP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed Collimator for 405 nm light</td>
			<td>Thorlabs</td>
			<td>F671SMA-405</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed collimator for 470 and 560 nm light</td>
			<td>Thorlabs</td>
			<td>F240SMA-532</td>
			<td></td>
		</tr>
		<tr>
			<td>Green emission filter</td>
			<td>Semrock</td>
			<td>FF01-520/35-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>High-Resolution USB 3.0 CMOS Camera</td>
			<td>Thorlabs</td>
			<td>DCC3260M</td>
			<td></td>
		</tr>
		<tr>
			<td>Light beam splitter</td>
			<td>Neurophotometrics</td>
			<td>SPLIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 425 nm Cutoff</td>
			<td>Thorlabs</td>
			<td>DMLP425R</td>
			<td></td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 495 nm Cutoff</td>
			<td>Semrock</td>
			<td>FF495-Di03</td>
			<td></td>
		</tr>
		<tr>
			<td>Metabond dental cement</td>
			<td>C&#38;B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M8 - M8 cable</td>
			<td>Doric Lenses</td>
			<td>Cable_M8-M8</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber cannulas</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Optic fiber Patchcords</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Red emission filter</td>
			<td>Semrock</td>
			<td>FF01-600/37-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>T7 LabJack</td>
			<td>LabJack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-cube LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 I/O Cable, Hirose 25, for DCC3240</td>
			<td>Thorlabs</td>
			<td>CAB-DCU-T3</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording neural activity in freely moving mice using fiber photometry

Source: Martianova, E., et al. <a href="https://app.jove.com/v/60278">Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals.</a> J. Vis. Exp. (2019).
This video demonstrates a method for recording neural activity in a freely moving mouse using fiber photometry. In this method, neurons in a specific brain region express a genetically encoded calcium indicator. A stimulus is applied to this brain region, and the stimulus-dependent change in fluorescence is measured to identify neural activity.

<img alt="Figure 1" src="/files/ftp_upload/22738/22738_Figure1.jpg" /> 
Figure 1: Fiber photometry schematic. (A) The excitation light from two LEDs (410 nm and 470 nm) passes through a series of filters and dichroic mirrors and produces an excitation spot at the working distance of the 20x objective. The light passes through either a single patch cord or bundled fibers (for multiple site recordings) connected to the implanted cannulas. The emitted fluorescence is co...

Recording Neural Activity in Freely Moving Mice Using Fiber Photometry

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Start with a freely moving mouse with an implanted optic fiber cannula.
Connect one end of a fiber-optic patch cord to the cannula and the other end to an excitation light source.
The mouse expresses a green fluorophore-tagged genetically encoded calcium indicator, or GECI, in the lateral hypothalamic area, or LHA neurons.
The implanted optic fiber contacts the lateral habenula, or LHb, region, where the LHA axons terminate.
Excite the GECI at its optimal excitation wavelength and record the baseline signal.
Now, apply repeated air puffs to the mouse to evoke an aversive stress response.
This stimulus triggers action potentials in LHA axon terminals, opening voltage-gated calcium channels and allowing calcium influx.
Intracellular calcium ions bind to the GECI, inducing a conformational change that increases green fluorescence emission.
Visualize the optical signal.
Increased fluorescence in response to air puffs in the mouse confirms the role of the LHA-LHb pathway in transmitting aversive signals.

recording-neural-activity-in-freely-moving-mice-using-fiber-photometry

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

257 次观看. 来源:Martianova， E. 等人。多纤维光度法记录自由移动动物的神经活动。J. Vis. Exp.（2019 年）。 该视频演示了一种使用光纤光度法记录自由移动的小鼠神经活动的方法。在这种方法中，特定大脑区域中的神经元表达遗传编码的钙指示剂。对该大脑区域施加刺激，并测量刺激依赖性的荧光变化以识别神经活动。 

视频: 使用纤维光度法记录自由移动小鼠的神经活动 - 概念

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

科研

JoVE Journal

神经科学

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.

来自多个小生物体的自动多模态刺激和同时神经元记录

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons ...

Recording Neural Activity in Freely Moving Mice Using Fiber Photometry

成績單

Recording Neural Activity in Freely Moving Mice Using Fiber Photometry